Egg production is an important economic trait in the commercial poultry industry. Ovarian follicle development plays a pivotal role in regulation of laying hen performance and reproductive physiology. However, the key genes and signaling pathways involved in the various-stages of laying hen follicular development remain poorly understood. In this study, transcriptomes of ovarian follicles at three developmental stages, the large white follicle (LWF), small yellow follicle (SYF), and large yellow follicle (LYF), were comparatively analyzed in hens with high (HR) and low (LR) egg-laying rates by RNA-sequencing. Eighteen cDNA libraries were constructed and a total of 236, 544, and 386 unigenes were significantly differentially expressed in the LWF, SYF, and LYF follicles of HR and LR hens, respectively. Among them, 47 co-transcribed differentially expressed genes (DEGs) in LWF and SYF, 68 co-expressed DEGs in SYF and LYF, and 54 co-expressed DEGs in LWF and LYF were mined. Thirteen co-expressed DEGs were found in LWF, SYF, and LYF follicles. Eighteen candidate genes, including P2RX1, CAB39L, BLK, CSMD3, GPR65, ADRB2, CSMD1, PLPP4, ATF3, PRLL, STMN3, RORB, PIK3R1, PERP1, ACSBG1, MRTO4, CDKN1A, and EDA2R were identified to be potentially related to egg production. Furthermore, Kyoto Encyclopedia of Genes and Genomes analysis indicated neuroactive ligand-receptor interaction, cell adhesion molecules, peroxisome proliferator-activated receptor pathway, and cAMP signaling pathway might elicit an important role in formation of egg-laying traits by influencing ovarian follicle development. This study represents the first transcriptome analysis of various-sized follicles between HR and LR hens. These results provide useful molecular evidence for elucidating the genetic mechanism underlying ovarian follicle development associated with egg production in chicken.

RAC1 belongs to the small G protein Rho subfamily and is implicated in regulating gene expression, cell proliferation and differentiation in mammals and humans; nevertheless, the function of RAC1 in growth and development of hen ovarian follicles is still unclear. This study sought to understand the biological effects of RAC1 on granulosa cell (GC) proliferation and differentiation of hen ovarian prehierarchical follicles. Firstly, our results showed expression levels of RAC1 mRNA in the follicles with diameters of 7.0–8.0 mm, 6.0–6.9 mm and 1.0–3.9 mm were greater than other follicles (p < 0.05). The RAC1 protein was mainly expressed in oocyte and its around GCs and stromal tissues of the prehierarchical follicles by immunohistochemistry. Further investigation revealed the RAC1 gene remarkably enhanced the mRNA and protein expression levels of FSHR (a marker of follicle selection), CCND2 (a marker of cell-cycle progression and GC differentiation), PCNA (a marker of GC proliferation), StAR and CYP11A1 (markers of GC differentiation and steroidogenesis) (p < 0.05). Furthermore, our data demonstrated siRNA interference of RAC1 significantly reduced GC proliferation (p < 0.05), while RAC1 gene overexpression enhanced GC proliferation in vitro (p < 0.05). Collectively, this study provided new evidence that the biological effects of RAC1 on GC proliferation, differentiation and steroidogenesis of chicken ovary follicles.

Transcription cofactors Vestigial like family (VGLL) members consisting of four homologs (VGLL1-4) are associated with cell growth and metastasis in mammals, among which VGLL1 gene has been documented to possess tumorigenic functions in various types of tumor, and VGLL4 acts as a new tumor suppressor; likewise several studies indicated that they potentially play a role in the regulation of ovary growth and function. However, the biological effects of chicken VGLL1 and VGLL4 on the proliferation, apoptosis, and steroidogenesis of the granulosa cells (GCs) during ovarian follicle development remain unknown now. This study found that VGLL1 and VGLL4 genes present divergent expression patterns of the transcripts in the GCs of various sized prehierarchical follicles (PFs) before follicle selection. Specific small interfering RNA (siRNA) was employed to elucidate the exact roles of VGLL1 and VGLL4 in regulating the PF development of the hen ovary. The results demonstrated that the mRNA expression levels of the steroidogenic-related enzyme steroidogenic acute regulatory protein (STAR) gene and the cell proliferation-related factors B-cell lymphoma-2 (BCL2), and cyclin D1 (CCND1) genes were significantly down-regulated in the cells with VGLL1 silence but remarkably up-regulated in the cells lacking VGLL4. Whereas the expression level of the cell apoptosis biomarker caspase-3 (CASP3) transcript was noticeably enhanced in the GCs without VGLL1 but significantly decreased in the GCs deprived of VGLL4. Further results showed that the siRNA-mediated silence of VGLL1 caused a significant increase in apoptosis with a reduction in the proliferation of GCs. Nevertheless, knockdown of VGLL4 resulted in a remarkable decrement in apoptosis but a memorable augment in proliferation of the GCs. Taken together, this study proved that VGLL1 promotes cell proliferation and steroidogenesis but inhibits apoptosis. In contrast, VGLL4 stimulates GC apoptosis while suppressing the GC proliferation and steroidogenesis in the hen ovarian follicles. We conluded that VGLL1 and VGLL4 affect oppositely the ovarian prehierarchical follicle development by the different regulatory manner in the GC proliferation and apoptosis of chicken ovary.

In a quest to find affordable, antibiotics free and readily available means of increasing broiler meat productivity, a number of natural feed additives have been tested and suggested. Garlic (Allium sativum) has emerged as a preferable alternative to synthetic feed additives. The aim of this study was to assess the effect of garlic as a feed additive on feed intake, weight gain, feed conversion ratio, and immune response. 300-day-old chicks were acclimatized for 2 weeks and then randomly assigned to 5 treatments (0.00%, 0.25%, 0.50%, 0.75%, and 1.00% garlic inclusion levels) with replicates. Mean weekly feed intake, weight gain, feed conversion ratio, and differential leucocyte counts (lymphocytes, heterophils, basophils, eosinophils, and monocytes) were analyzed for treatment effects. There were significant differences among the means across all treatments for all dependent variables except mean weekly body weight gain at 2 weeks. Garlic supplementation significantly influenced body weight from three to six weeks of age, with the control group (0.00%) consistently exhibiting the lowest body weight. At six weeks, body weight was highest at 0.75%, followed by 1.00%, 0.50%, and 0.25% garlic inclusion levels, demonstrating the positive impact of dietary garlic on growth performance. It was also observed that garlic supplementation led to increases in the basophil and monocyte counts, suggesting a bolstered immune system.